The invention relates to instrumentation and methods for detecting light. In particular, the invention relates to a versatile, sensitive, high-throughput screening apparatus that quantifies light transmitted from an assay site.
High-throughput screening instruments are critical tools in the pharmaceutical research industry and in the process of discovering and developing new drugs. The drug discovery process involves synthesis and testing, or screening, of candidate drug compounds against a target. A candidate drug compound is a molecule that might mediate a disease by its effect on a target. A target is a biological molecule, such as an enzyme, receptor, other protein, or nucleic acid, that is believed to play a role in the onset or progression of a disease or a symptom of a disease. FIG. 1 shows stages of the drug discovery process, which include target identification, compound synthesis, assay development, screening, secondary screening of hits, and lead compound screening, or optimization, and finally clinical evaluation.
Targets are identified based on their anticipated role in the progression or prevention of a disease. Until recently, scientists using conventional methods had identified only a few hundred targets, many of which have not been comprehensively screened. Recent developments in molecular biology and genomics have led to a dramatic increase in the number of targets available for drug discovery research.
After a target is selected, a library of compounds is selected to screen against the target. Compounds historically have been obtained from natural sources or synthesized one at a time. Compound libraries were compiled over decades by pharmaceutical companies using conventional synthesis techniques. Recent advances in combinatorial chemistry and other chemical synthesis techniques, as well as licensing arrangements, have enabled industrial and academic groups greatly to increase the supply and diversity of compounds available for screening against targets. As a result, many researchers are gaining access to libraries of hundreds of thousands of compounds in months rather than years.
Following selection of a target and compound library, the compounds must be screened to determine their effect on the target, if any. A compound that has an effect on the target is defined as a hit. A greater number of compounds screened against a given target results in a higher statistical probability that a hit will be identified.
Prior to screening compounds against a target, a biological test or assay must be developed. An assay is a combination of reagents that is used to measure the effect of a compound on the activity of a target. Assay development involves selection and optimization of an assay that will measure performance of a compound against the selected target. Assays are broadly classified as either biochemical or cellular. Biochemical assays usually are performed with purified molecular targets, which generally have certain advantages, such as speed, convenience, simplicity, and specificity. Cellular assays are performed with living cells, which may sacrifice speed and simplicity, but which may provide more biologically relevant information. Researchers use both biochemical and cellular assays in drug discovery research.
Biochemical and cellular assays may use a variety of detection modalities, including photoluminescence, chemiluminescence, and absorbance. Photoluminescence and chemiluminescence assays involve determining the amount of light that is emitted from excited electronic states created by absorption of light and certain chemical reactions, respectively. Absorbance assays involve determining the amount of light that is transmitted through a composition relative to the amount of light incident on the composition.
Each detection modality may use a variety of equipment. For example, photoluminescence assays typically employ at least a light source, detector, and filter; absorbance assays typically employ at least a light source and detector; and chemiluminescence assays typically employ at least a detector. Moreover, the type of light source, detector, and/or filter employed typically varies even within a single detection modality. For example, among photoluminescence assays, photoluminescence intensity and steady-state photoluminescence polarization assays may use a continuous light source, and time-resolved photoluminescence polarization assays may use a time-varying light source.
Adding to this variability, the types of assays that are desired for high-throughput screening are evolving constantly. As new assays are developed in research laboratories, tested, and published in literature or presented at scientific conferences, new assays become popular and many become available commercially. New analytical equipment may be required to support the most popular commercially available assays.
After selection of a target, compound library, and assay, assays are run to identify promising compound candidates or hits. Once a compound is identified as a hit, a number of secondary screens are performed to evaluate its potency and specificity for the intended target. This cycle of repeated screening continues until a small number of lead compounds are selected. The lead compounds are optimized by further screening. Optimized lead compounds with the greatest therapeutic potential may be selected for clinical evaluation.
Due to the recent dramatic increase in the number of available compounds and targets, a bottleneck has resulted at the screening stage of the drug discovery process. Historically, screening has been a manual, time-consuming process. Recently, screening has become more automated, and standard high-density containers known as microplates have been developed to facilitate automated screening. Microplates are substantially rectilinear containers that include a plurality of sample wells for containing a plurality of samples. Ninety-six-well microplate formats have been and still are commonly used throughout the high-throughput screening industry. However, some high-throughput screening laboratories are using 384- and 768-well plates, and some laboratories are experimenting with 1536-, 3456-, and 9600-well microplates.
FIG. 2 shows a stack of overlapping microplates with various well densities. Plate 30 has 96 wells. Plate 32 has 384 wells. Plate 34 has 1536 wells. Plate 36 has 3456 wells. Plate 38 has 9600 wells. FIG. 2 illustrates the substantial differences in well dimensions and densities that may be used in high-throughput screening assays. Many analyzers are not flexible enough to read microplates having different numbers of wells, such that it currently may be necessary to provide different analyzers for different modes of analysis. Moreover, many analyzers are not sensitive or accurate enough to read results from the smaller wells associated with the higher-density microplates. Inadequate sensitivity may result in missed hits, limited research capabilities, increased costs of compounds, assays, and reagents, and lower throughput.
Screening an increasing number of compounds against an increasing number of targets requires a system that can operate with a high degree of automation, analytical flexibility, and speed. In particular, because high-throughput applications may involve repeating the same operations hundreds of thousands of times, even the smallest shortcomings are greatly magnified. Current screening systems operate with various degrees of automation. Automation, from sample dispensing to data collection, enables round-the-clock operation, thereby increasing the screening rate. Automated high-throughput screening systems usually include combinations of assay analyzers, liquid handling systems, robotics, computers for data management, reagents and assay kits, and microplates.
Most analyzers in use today are not designed specifically for high-throughput screening purposes. They are difficult and expensive to integrate into a high-throughput screening environment. Even after the analyzer is integrated into the high-throughput screening environment, there often are many problems, including increased probability of system failures, loss of data, time delays, and loss of costly compounds and reagents.
In addition, most analyzers in use today offer only a single assay modality, such as absorbance or chemiluminescence, or a limited set of modalities with non-optimum performance. To perform assays using different detection modes, researchers generally must switch single-mode analyzers and reconfigure the high-throughput screening line. Alternatively, researchers may set up the high-throughput screening line with multiple single-mode analyzers, which often results in critical space constraints.
Thus, prior detection devices generally have not recognized the need to provide analytic flexibility and high performance for assay development as well as ease of use and smooth automation interface for the high-throughput screening laboratory. A real need exists for a versatile, sensitive, high-throughput screening apparatus that can handle multiple detection modalities and wide ranges of sample volumes and variations in container material, geometry, size, and density format while reliably maintaining a high level of sensitivity.
The present invention provides an apparatus and method for detecting light transmitted from a composition. The apparatus and method may emphasize plural light sources and/or detectors. The apparatus and method also may emphasize top/bottom illumination and/or detection.
In an embodiment emphasizing plural light sources, the apparatus includes (1) a stage for supporting a composition at an examination site, (2) at least two different light sources and a first optical relay structure that directs light from one of the light sources toward the composition, (3) a detector and a second optical relay structure that directs light from the composition toward the detector, and (4) a first switch mechanism that alters alignment of the first optical relay structure from one of the light sources to another of the light sources, so that different light sources can be selected and directed toward the examination site for different applications.
In an embodiment emphasizing plural detectors, the apparatus includes (1) a stage for supporting a composition at an examination site, (2) a light source and a first optical relay structure that directs light from the light source toward the composition, (3) at least two detectors and a second optical relay structure that directs light from the composition to one of the detectors, and (4) a first switch mechanism that alters alignment of the second optical relay structure from one of the detectors to another of the detectors, so that different detectors can be selected for different applications.
In an embodiment emphasizing top/bottom illumination, the apparatus includes (1) a stage for supporting a composition at an examination site, the examination site having a top side and a bottom side, (2) at least one light source and a first optical relay structure defining a first optical path directed toward the top side of the examination site and a second optical path directed toward the bottom side of the examination site, (3) at least one detector and a second optical relay structure that directs light from the composition toward the detector, and (4) a first switch mechanism that alters alignment of the light source from one of the optical paths to the other optical path.
In an embodiment emphasizing top/bottom detection, the apparatus includes (1) a stage for supporting a composition at an examination site having a top side and a bottom side, (2) at least one light source and a first optical relay structure that directs light from the light source toward the composition, (3) at least one detector and a second optical relay structure defining a first optical path directed toward the top side of the examination site and a second optical path directed toward the bottom side of the examination site, and (4) a first switch mechanism that alters alignment of the detector from one of the optical paths to the other optical path.
In yet other embodiments, light sources and detectors are replaced with adjacent compartments for light sources and detectors.
The apparatuses described above further may include (1) additional light sources and detectors, (2) controllers preprogrammed to activate the switch mechanisms for selecting light sources and detectors for particular assays, (3) bar code readers for further automating the controllers, (4) filter alignment mechanisms for aligning filters with light sources and detectors, (5) shuttles for aligning the optical relay structures, and (6) automated registration devices for facilitating successive analysis of multiple compositions. The optical relay structures further may include optical paths connecting light sources and detectors to top and bottom sides of the examination site to permit (1) top-illumination and top-detection, (2) top-illumination and bottom-detection, (3) bottom-illumination and top-detection, and (4) bottom-illumination and bottom-detection. Preferred light sources include high-intensity, high-color temperature arc lamps, and preferred detectors include photomultiplier tubes.
The present invention also provides methods of detecting light transmitted from a composition.
In an embodiment emphasizing plural light sources, the method includes (1) providing a plurality of light sources, at least one detector, and an optical relay structure in a light detection instrument, wherein the optical relay structure directs light from one of the light sources toward a composition at an examination site, (2) selecting one of the light sources using a first switch mechanism that alters alignment of the optical relay structure from one of the light sources to another of the light sources, (3) relaying light from the selected light source through the optical relay structure to the composition, and (4) detecting light transmitted from the composition.
In an embodiment emphasizing plural detectors, the method includes (1) providing at least one light source, a plurality of detectors, and an optical relay structure in a light detection instrument, wherein the optical relay structure directs light from a composition at an examination site toward one of the detectors, (2) selecting one of the detectors using a first switch mechanism that alters alignment of the first optical relay structure from one of the detectors to another of the detectors, (3) illuminating the composition, and (4) relaying light from the composition through the optical relay structure to the selected detector.
The methods described above further may involve selecting among both light sources and detectors, and/or among top/bottom illumination and detection.
The nature of the invention will be understood more readily after consideration of the drawings and the detailed description of the preferred embodiment that follow.